Corticotropin-releasing hormone stimulates expression of leptin, 11beta-HSD2 and syncytin-1 in primary human trophoblasts.

Fahlbusch et al. Reproductive Biology and Endocrinology 2012, 10:80
http://www.rbej.eom/content/1 0/1 /80

REPRODUCTIVE BIOLOGY
AND ENDOCRINOLOGY

RESEARCH Open Access

Corticotropin-releasing hormone stimulates
expression of leptin, 11beta-HSD2 and syncytin-1
in primary human trophoblasts

Fabian B Fahlbusch 1 , Matthias Ruebner 2 , Gudrun Volkert 1 , Ramona Offergeld 1 , Andrea Hartner 1 ,
Carlos Menendez-Castro 1 , Reiner Strick 2 , Manfred Rauh 1 , Wolfgang Rascher 1 and Jorg Dbtsch 3

Abstract

Background: The placental syncytiotrophoblast is the major source of maternal plasma corticotropin-releasing
hormone (CRH) in the second half of pregnancy. Placental CRH exerts multiple functions in the maternal organism:
It induces the adrenal secretion of Cortisol via the stimulation of adrenocorticotropic hormone, regulates the
timing of birth via its actions in the myometrium and inhibits the invasion of extravillous trophoblast cells in vitro.
However, the auto- and paracrine actions of CRH on the syncytiotrophoblast itself are unknown. Intrauterine
growth restriction (IUGR) is accompanied by an increase in placental CRH, which could be of pathophysiological
relevance for the dysregulation in syncytialisation seen in IUGR placentas.

Methods: We aimed to determine the effect of CRH on isolated primary trophoblastic cells in vitro. After CRH
stimulation the trophoblast syncytialisation rate was monitored via syncytin-1 gene expression and beta-hCG
(beta-human chorionic gonadotropine) ELISA in culture supernatant. The expression of the IUGR marker genes
leptin and 1 1 beta-hydroxysteroid dehydrogenase 2 (1 1beta-HSD2) was measured continuously over a period
of 72 h. We hypothesized that CRH might attenuate syncytialisation, induce leptin, and reduce 1 1beta-HSD2
expression in primary villous trophoblasts, which are known features of IUGR.

Results: CRH did not influence the differentiation of isolated trophoblasts into functional syncytium as determined
by beta-hCG secretion, albeit inducing syncytin-1 expression. Following syncytialisation, CRH treatment significantly
increased leptin and 1 1beta-HSD2 expression, as well as leptin secretion into culture supernatant after 48 h.

Conclusion: The relevance of CRH for placental physiology is underlined by the present in vitro study. The
induction of leptin and 1 1beta-HSD2 in the syncytiotrophoblast by CRH might promote fetal nutrient supply and
placental corticosteroid metabolism in the phase before labour induction.

Keywords: CRH, leptin, 1 1beta-HSD2, Syncytin-1, Trophoblast, Syncytiotrophoblast, Placenta

Background

As part of the neuroendocrine system, the hypothalamo-
pituitary-adrenal axis controls a wide range of body func-
tions in humans. Hypothalamic corticotropin-releasing
hormone (CRH) acts via its two receptors CRH-R1 and
CRH-R2 to control stress reaction, autonomic functions,
behavioural response, appetite, metabolism and the
immune system.

* Correspondence: fabian.fahlbusch@uk-erlangen.de
'Department of Pediatrics and Adolescent Medicine, University of
Erlangen-Nurnberg, Erlangen, Germany

Full list of author information is available at the end of the article

Bio Med Central

Since its discovery in placental extracts in 1982 [1] it
has become evident, that CRH and the related peptide
urocortin [2] also exert important functional roles in
human reproductive physiology [3,4]. CRH and its
receptors are present in ovaries [5], endometrium [6],
decidua [7], myometrium [8] and in the placenta (syncy-
tiotrophoblast, chorion and amnion) [9,10]. The placen-
tal syncytiotrophoblast is a major source of plasma CRH
in the maternal circulation in the second half of preg-
nancy [11]. Multiple isoforms of the CRH receptors
CRH-R1 and CRH-R2 were identified in the placental
trophoblast [9,10] and myometrium [12,13] throughout

© 2012 Fahlbusch et al.; licensee BioMed Central ftd. This is an Open Access article distributed under the terms of the
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gestation. Hence differential local effects on fetal and
maternal intra-uterine tissues are conceivable. The
effects of placental CRH have been intensively studied
indirectly via the action of the Cortisol proxys leptin and
11|3-HSD2 [14,15] and directly at the placental bed,
where it plays an important role in the timing of birth
in humans [16]. CRH interacts with progesterone to
enhance the contractile response of the myometrium
[16,17] and regulates the vascular tonus in the fetopla-
cental circulation through the nitric oxide (NO)/cGMP
pathway [18,19]. Bamberger et al. [20] have recently
shown, that CRH inhibits extravillous trophoblast (EVT)
invasion by decreasing the expression of CEACAM1 via
signalling through CRH-R1. There is growing evidence
that a dysregulation of spiral artery invasion by EVT in
the first trimester is a process contributing to the vascu-
lar resistance observed in the pregnancy complications
preeclampsia and intrauterine growth restriction (IUGR)
in late pregnancy [21,22]. In line with this finding, we
and others have previously shown that placental CRH
expression and CRH in maternal plasma are significantly
elevated in IUGR [23-26]. IUGR is further pathophysio-
logically characterized by a reduction in trophoblastic
syncytialisation rate [27], increased leptin [28] and
reduced llp-HSD2 [29] expression.

Although it is known that the syncytiotrophoblast is a
major source of CRH in the second half of pregnancy,
the role of CRH on the process of cytotrophoblastic
syncytialisation and on endocrine hormone regulation
in the syncytiotrophoblast is unknown so far.

To further investigate local actions of CRH on tropho-
blast function, we sought to determine its influence
on the syncytialisation of isolated primary villous
trophoblastic cells and on the expression of leptin and
11P-HSD2. We found that CRH induced leptin and 110-
HSD2 expression, without affecting syncytialisation of
trophoblastic cells.

Methods

Placental collection and tissue culture

Six term placentas from women with singleton uncom-
plicated pregnancies were collected immediately after
placental delivery. Elective caesarean section delivery
was performed and birth weight was >10 th percentile
according to Voigt et al. [30]. Primary human cytotro-
phoblasts were isolated from the placentas using the
established trypsine-DNAse-dispase/percoll method as
initially described by Kliman et al. [31], with additional
previously published modifications [32,33]. The purity
of trophoblastic cells was routinely controled by mul-
tiple FACS analysis (FACSCalibur, BD Biosciences), as
described previously [33], providing at least 90% cytotro-
phoblasts. In short, we determined that 10-13.3% of the
fractions were HLA-A,B,C + (mainly mononuclear blood

cells and fibroblasts) and 86.6-90% HLA-A,B,C negative.
Additionally, fractionated cells were 2.4-4.5% CD45+
(mononuclear blood cells) and 95.5-97.6% CK7+ (epi-
thelial marker). Antibodies used: CK7/PE (clone 5 F282),
Santa Cruz Bio., Heidelberg, Germany (1:20); HLA-A,B,
C/PE (cloneW6/32), Biolegend, Uithoorn, Netherlands
(1:10); CD45/FITC, Miltenyi Biotec, Berg. Gladbach,
Germany (1:10). Hence 86.6-90% of the fractionated
cells were trophoblastic cells and 10-13.3% non-
trophoblastic cells. Multinucleated fractured syncytial
fragments were identified via their DNA-content using
propidium iodide staining (Sigma-Aldrich Chemie,
Munich, Germany; 50 ug/ml), specific for DNA content.
All fractured syncytial cellular fragments, non-adherent
cells and debris were removed initially after 4 h and
then every 24 h with a medium change [34]. Cells were
subsequently seeded into 6-well Falcon plates (Becton
Dickinson, Heidelberg, Germany) at a density of 3x10 s
cells/cm and maintained in Earl's medium 199 (PAA
Laboratories, Linz, Austria) supplemented with 10%
fetal calf serum (PAA Laboratories), 20 mM Hepes
(Sigma-Aldrich), 0.5 mM L-glutamine (Gibco Invitro-
gen, Karlsruhe, Germany), penicillin (10 U/ml), strepto-
mycin (10 mg/ml), and fungizone (0.25 mg/ml) (Sigma-
Aldrich, Gibco Invitrogen, respectively). Cultures were
grown at 37 °C under normoxia with 95% air, 5% CO2
in a humidified atmosphere using a Forma Scientific
incubator (Fisher Scientific, Schwerte, Germany) as
described in detail previously [33]. After incubation for
24 h, trophoblastic cells were stimulated with 0.5, 1.0
and 2.0 ug/ml (equivalent to 100, 200, 400 nM, respect-
ively) CRH (Bachem, Weil am Rhein, Germany) for 6,
12, 24, 48 and 72 h. This range of concentrations was
chosen, as it has been described to exert biological
effects on trophoblasts [20]. Our pilot study showed no
difference of repetitive stimulation vs. single application
of CRH to the cell culture, with significant changes
in gene expression between vehicle and CHR-treated
groups starting with 48 h (1.0 and 2.0 ug/ml CRH) for
the analysed genes. 72 h were chosen as the maximum
observational period, because our previous experiments
have shown that cytotrophoblast viablitiy steadily decreases
afterwards. Hence for illustration of group differences the
time-points of 48 h and 72 h and CRH concentrations of
1.0 ug/ml and 2.0 u/ml are displayed in the results section
only. Cultured trophoblasts as well as culture supernatants
were collected at the time-points described above, snap
frozen and stored at -80°C until further processing. All
experiments were assayed in triplicate and were repeated
using cells from different placentas.

Ethics

The study was reviewed and approved by the Ethics
Committee of the Medical Faculty of the University of

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Erlangen-Niirnberg (#2625 02/28/02). Written informed
consent was obtained from all subjects.

RNA isolation and reverse transcription-polymerase chain
reaction (RT-PCR)

Total RNA was isolated from primary human tropho-
blasts using TRIzol reagent (Gibco Invitrogen) as recom-
mended by the manufacturer. RNA was quantified by
absorbance at 260 nm and the quality of RNA was con-
firmed using a 1% agarose gel. After DNase treatment,
1.0 mg RNA was transcribed into cDNA using M-MLV-
RT (Promega, Madison, WI, USA) and Oligo dT-primer
(MWG-Biotech AG, Ebersberg, Germany). DNase treat-
ment and cDNA synthesis were carried out as previously
[33,35].

SYBR-Green based real-time PCR (2 ~^ CT - method)
As previously described [14] the mRNA expression of
leptin, 11|3-HSD2, Syncytin-1, CRH-Rl and CRH-R2
were quantified by normalising to the house-keeping
gene hypoxanthine guanine phosphoribosyl transferase
(HPRT) and confirmed with rl8S as a second house-
keeper, yielding the same results. Commercial reagents
(Absolute Blue SYBR Green master mix, ABgene, UK)
and conditions were applied according to the manufac-
turer's protocol. Serial dilutions of one of the samples
served as reference providing relative quantification of
the unknown samples. Sequences of primers and probes
are listed in Table 1.

Determination of p-hCG, leptin and LDH concentration in
the supernatant

The concentration of (3-human chorionic gonadotropine
(|3-hCG) in the supernatants of trophoblastic cells was
determined by the use of an UniCel Dxl 600 Access Im-
munoassay System (Beckman Coulter, Krefeld, Germany).
The concentration of leptin in the supernatants of

Table 1 Primer sequences

Forward (5 '-3')

Reverse (5 '-3')

HPRT

r18S

CCGGCTCCGTTATGGC

GCAATTATTGCCCATG-
AACG

GGTCATAACCTGGTTCA-
TCATCA

GGCCTCACTAAACCAT-
CCAA

trophoblastic cells was determined by the RayBio®
Human Leptin ELISA Kit (RayBiotech, Norcross, GA,
USA) according to the manufacturer's instructions. Lac-
tate dehydrogenase (LDH) concentrations were obtained
spectrophotometrically [36] by the In Vitro Toxicology
Assay Kit Lactate Dehydrogenase based (TOX-7, Sigma-
Aldrich). Determination of culture supernatant protein
content for protein normalisation was performed with
Pierce bicinchoninic acid (BCA) Protein Assay Reagent
(Thermo Fisher Scientific, Bonn, Germany). All measure-
ments were assayed in triplicate. Analysis of the results
was performed using Ascent Software v2.6 for Multiscan
photometer (Thermo Fisher Scientific).

Statistical analysis

Results were expressed as mean ± standard error of the
mean (SEM). Differences were assessed using the non-
parametric Mann- Whitney U test provided with SPSS
statistic software (vl9.001, IBM, Ehningen, Germany). A
p-value of <0.05 was considered significant.

Results

Assessment of primary trophoblastic cell viability and
functionality

At the sequential experimental time-points LDH was
assessed spectrophotometrically using the trophoblastic
cell supernatants. There was no significant increase
observed during trophoblastic cell culture, nor were
group differences detected between unstimulated con-
trols and CRH-treated trophoblast cells in terms of via-
bility (Table 2), ruling out a contamination of the
supernatant with intracellular (3-hCG as a consequence
of cell lysis. The |3-hCG secretion is a valid parameter of
trophoblast syncytialisation rate [31]. The [3-hCG con-
tent of the trophoblastic cell supernatant, as assessed
by ELISA, increased continuously over the time-points
investigated in both experimental groups. After 24 h of
culture, the increase became significant (p < 0.01) evi-
dencing the progression of syncytialisation of the
trophoblastic cells (data not shown). Compared to 6 h,
both vehicle and CRH treated primary trophoblastic cells

Table 2 LDH absorbance in the culture medium of human
trophoblastic cells with and without CRH (1 ug/ml)
stimulation

Leptin

ACAATTGTCAGCAGGA-

TCCAAACCGGTGAGTT-

Time (hours)

Absorbance levels (rel. units)

p-value

TCAATGAC

TCTGT

Vehicle

CRH

1 1(i-HSD2

CATCACCGGCTGTGAG-

CGGCAGCCGCATGTTAG

mean

SEM

mean

SEM

TCTG

6

0.021

0.006

0.022

0.013

ns

CRH-Rl
alpha

CTACATGCTGTTCTTCG-
TCAATCC

GGCAGAAGGGAGCTGGAA

12

0.036

0.005

0.032

0.001

ns

CRH-R2

TCCAGTACAGGAAGG-

GGAGTTGAAATAGATGAA-

24

0.026

0.017

-0.004

0.003

ns

CAGTGAA

CATGATCTG

48

0.033

0.004

0.019

0.001

ns

Syncytin- 1

ATGGAGCCCAAGATGCAG

AGATCGTGGGCTAGCAG

72

0.025

0.005

0.029

0.006

ns

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showed a significant increase of (3-hCG protein content
in the supernatant at 48 h (p < 0.01) and more signifi-
cantly at 72 h (p < 0.001, data not shown). Stimulation
with CRH (1.0 and 2.0 ug/ml) did not influence
the amount of [3-hCG in the supernatant at 48 and 72 h
(Figure 1), indicating that CRH does not alter maturation

of trophoblastic cells in vitro. Additionally we measured
syncytin-1 (Synl) expression, as previously described
[33,35]. Synl is essential for mediating trophoblast cell
fusion events [37]. Synl expression was significantly
induced at 48 h by CRH in a dose-dependent manner
(1.0 ug < 2.0 ug, p < 0.029 for both, Figure 1). At 72 h the

48h

control I.OpgCRH 2.0pg CRH

control I.OpgCRH 2.0pg CRH

control I.OpgCRH 2.0pg CRH

72h

control 1.0|jgCRH 2.0|jg CRH

control I.OpgCRH 2.0(jg CRH

control 1.0|igCRH 2.0pg CRH

I Leptln
■ Leptin P

iSynl

a CRH-R1
■ CRH-R2
11CHSD2

Figure 1 Overview of gene expression profiles and results of protein detection. Overview of gene expression profiles (RT-PCR) and results
of protein detection (ELISA) in cell culture supernatant of vehicle and corticotropin-releasing hormone (CRH) (1.0 and 2.0 ug/ml) treated
trophoblasts at 48 and 72 h. Top row: Leptin gene expression (Leptin, blue bars), Leptin protein secretion (Leptin P, red bars). Middle row:
Syncytin-1 (Synl) (blue bars), (5-hCG (red bars). Bottom row: CRH-R1 (blue bars), CRH-R2 (red bars), 1 1 (3-H5D2 (green bars). Displayed are values
relative to the control value at the designated time-point as mean ± SEM, * = p < 0.05.

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stimulative effect of CRH on Synl expression had sub-
sided (Figure 1).

At 72 h the stimulatory effect of CRH had subsided for
both 1.0 and 2.0 ug/ml CRH concentrations.

Leptin expression

Previous experiments have shown a close relation of
trophoblast leptin expression to leptin secretion [14].
Leptin expression increased with culture time of tropho-
blastic cells, irrespective of the stimulation with CRH.
This increase was significant after 12 h of culture
and peaked after 24 h (Figure 2; p<0.05). While leptin
expression levels in the unstimulated control group
declined after 48 h, CRH-treated primary trophoblastic
cells showed a more sustained induction of leptin
expression (Figure 2). At this time-point, leptin expres-
sion was significantly higher in the trophoblastic cells
after stimulation with 1.0 and 2.0 ug/ml CRH compared
to unstimulated controls (p < 0.05, Figure 1 and Figure 2).
After 72 h the leptin expression had returned to a basal
level in both groups alike. Leptin protein expression
closely matches expressional changes following CRH
stimulation (Figure 1). At 48 h CRH dose-dependently
increased in leptin secretion into trophoblast culture
supernatant. The stimulatory effect of 1.0 ug/ml CRH on
trophoblast leptin secretion lasted for 48 h (p < 0.029).
After 2.0 ug/ml CRH stimulation, there was still a sig-
nificant (p < 0.002) induction in leptin secretion detect-
able at 72 h (Figure 1).

11(1-HSD2 expression

Stimulation of trophoblastic cells with 2.0 ug/ml CRH
significantly (p < 0.029) induced 116-HSD-2 expression
at 48 h (Figure 1). Stimulation of trophoblastic cells with
1.0 ug/ml CRH did not significantly alter llfi-HSD-2 ex-
pression during the observational period (Figure 1).

CRH receptor expression

CRH-R2 gene expression was low (Q: 32.8 ± 0.35 SEM)
in cultured human trophoblasts throughout the duration
of the experiment, while CRH-R1 was readily detectable
(Q: 24.89 ±0.21 SEM). CRH treatment at a concentra-
tion of 2.0 ug/ml resulted in an increased CRH-R1 and
CRH-R2 expression in these cells (5.05 ±1.33 SEM,
p = 0.029; 4.55 ± 2.05 SEM, p = 0.029, respectively). Inter-
estingly, at 72 h CRH-R1 and CRH-R2 expression levels
following CRH treatment (1.0 and 2.0 ug/ml) were not
different to expression levels in vehicle treated controls.

Discussion

In the present study, we aimed to clarify the influence of
corticotropin-releasing hormone (CRH) exposure on the
syncytialisation rate of isolated primary villous tropho-
blastic cells. Moreover, we investigated whether CRH
induces alterations in gene expression of specific endo-
crine placental regulators in vitro. Our results showed a
significantly higher leptin expression in trophoblastic
cells, which concomitantly resulted in a significant
induction of leptin protein secretion into the supernatant
after 48 h of CRH stimulation, compared to unstimu-
lated control cells. Moreover, 11|3-HSD2 expression was
dose-dependendy induced by 2.0 ug/ml CRH after 48 h.
Formation of a functional syncytiotrophoblast occurred
after 24 h in the CRH-stimulated and the control group
to the same degree, as determined by increasing p-hCG
secretion without a concomitant increase of cell lysis
reflected by constant LDH levels in the supernatant
throughout the experiment [31]. Syncytin-1 expression,
a key regulator gene of trophoblast syncytialisation

I vehicle
I CRH

Figure 2 Leptin expression in human trophoblastic cells: Vehicle vs. CRH (1.0 ug/ml) stimulation. A significant (p<0.05) increase in relative
leptin gene expression was observed after 12 h in both groups. CRH treatment significantly (p<0.05, circle) increased leptin expression above
control levels after 48 h. Gene expression is related to the housekeeping gene HPRT. Displayed are means ± SEM, *p < 0.05, **p < 0.01 .

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[33,35,37], was dose-dependently induced after 48 h of
CRH stimulation only.

The finding that leptin expression increases with the
progression of trophoblast syncytialisation in both
groups is in line with results from Ashworth et al. [38],
who showed that placental leptin expression is an exclu-
sive feature of the syncytiotrophoblast (SCT), with a
reduced leptin expression in undifferentiated cytotro-
phoblasts. Likewise, CRH was described as a syncytical
peptide [39]. CRH-treated and vehicle stimulated tro-
phoblasts showed an equal increase of fi-hCG concen-
tration in the culture supernatant, while cell lysis was
low and not different (as determined by LDH). This
finding is indicative that the rate of trophoblast syncytia-
lisation gradually increased over time in both groups
[40]. The induction of leptin expression after 24 h of
culture of CRH- and vehicle treated trophoblastic cells
can therefore be attributed to trophoblastic differenti-
ation into functional syncytium. The significant differ-
ence in leptin gene expression in CRH-treated and
vehicle treated control groups at 48 h and the induction
of leptin protein secretion into the cell culture super-
natant, however, do not seem to be solely an indirect
effect of syncytialisation, as fi-hCG levels of CRH-
treated trophoblastic cells were not significantly different
from the fi-hCG levels of vehicle treated controls. Hence
the significant increase in leptin expression and secre-
tion of stimulated cells at 48 h rather seems to be a dir-
ect effect of CRH, possibly via activation of CRH-Rloc,
which was readily detectable in trophoblasts [10,41] and
towards which CRH shows a ten times higher affinity as
compared to CRH-R2 [42], whose expression was signifi-
cantly lower. The fact that we were able to detect CRH-
Rla and CRH-R2 in vitro does not rule out the possibil-
ity of CRH signalling via its other known placental iso-
forms that were described in vivo [10,41]. Interestingly
Karteris et al. found significant levels of receptor
hybridization foremost in syncytiotrophoblast and to a
lesser extend in scattered cytotrophoblasts, which is sup-
ported by almost exclusive binding of [ 125 I]CRH to puri-
fied syncytiotrophoblast [43]. This could suggest CRH
might rather excert its functions after spontaneous syn-
cytialisation of cytotrophoblasts in vitro than on single
cytotrophoblasts.

To address the functional relevance of our findings
studies with CRH antagonists, such as CRH antagonists
antalarmin (CRH-R1) and antisauvagine (CRH-R2) would
be needed.

We found that CRH (2 ug/ml) dose-dependently
induced CRH -Rla and -R2 gene expression at 48 h
(Figure 1, p < 0.029). The stimulatory effect of CRH on
leptin expression was abolished at 72 h. CRH-R
internalization could be a possible mechanism to explain
this phenomenon, as previously described by others

[44,45]. CRH-R internalization could also account for
the absence of an expressional effect on the studied tar-
get genes following repeated CRH stimulation in our
pilot study. Such an induction of CRH-R expression
levels following exposure to higher doses of CRH might
optimize CRH signal transduction. Interestingly, higher
concentrations of CRH (2.0 ug/ml) also significantly
induced 118-HSD2 expression at 48 h (Figure 1,
p < 0.029), while 1.0 ug/ml showed no such effect. Hence
higher levels of auto-/paracrine CRH might hypothetic-
ally prepare the syncytium to cope with a CRH-triggered
increase of maternal Cortisol more efficiently.

The above findings regarding leptin are in line with
findings from our previous experiments showing a close
relationship of trophoblast leptin expression and secretion
rate in vitro following dexamethasone stimulation [14].
Interestingly, dexamethasone stimulation (10 uM) pro-
duced a more pronounced leptin secretion (~120 pg/ml
at 72 h) when compared to CRH (1.0 and 2.0 ug/ml)
stimulation (-56-61 pg/ml at 72 h, respectively). However,
dexamethasone clearly induced fi-hCG secretion. Hence
in contrast to CRH the effect of dexamethasone on leptin
secretion seems to be partly attributable to an increased
rate of trophoblast differentiation and maturation, as also
seen by Audette et al. [46]. They were also able to dem-
onstrate a trend to Synl induction in placental explants
following dexamethasone treatment.

The fact, that we found an increase in Synl expression
following CRH stimulation without a concomitant in-
crease in fi-hCG might point to a differential regulation
of the two genes. A common pathway for both Synl and
15-hCG stimulation is the forskolin triggered induction
of cAMP [47]: An activation of adenyl cyclase (AC)
raises intracellular cAMP levels and leads to PKA activa-
tion via interactions with AKAPs and downstream phos-
phorylation of p38MAPK and ERK1/2. Accordingly,
CRH was found to induce cAMP in human endomet-
rium via CRHR1 triggered protein-kinase A (PKA) [48]
and we recently found that Synl is induced via the
cAMP pathway in endometrial carcinoma [49]. However,
as fi-hCG was not induced, CRH might either use alter-
native signalling pathways, such as signalling via PKC
[45,50], or the detected Synl expression might come
from a source other than the cytotrophoblast. However
our cytotrophoblast isolation is ~90% pure. Thus, we
cannot exclude possible minor fractions of extravillous
trophoblasts (EVT) in our cell culture. EVT express
CRHR1 [51] and show Synl expression [20,52]. There-
fore, we cannot completely rule out that the Synl induc-
tion measured is derived from EVT. But due to their
extremely small fraction in the isolation, a six-fold
increase in Synl expression by EVT following CRH-
stimulation seems rather unlikely. Another explanation
could be, that CRH fosters maintenance fusion events

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instead of functional fusion processes, that would be
reflected in fi-hCG secretion. Importantly, Synl is related
to trophoblast processes beyond its fusogenic nature.
Possible functions of the syncytin proteins are suppres-
sion of the maternal immune response against the devel-
oping fetus [53] and induction of placental immunity
against vertical transmission of retroviral infections [54].
We observed a stimulatory effect of CRH (2.0 ug/ml
after 48 h) on the expression of 1113-HSD2 in primary
cultured cytotrophoblasts. Like leptin, the induction of
11(3-HSD2 by CRH subsided at 72 h, possibly due to
CRH-receptor internalization, as discussed above. In a
previous study using the same in vitro setup, we were
able to show that dexamethasone (10 uM) similarly sti-
mulates both leptin and 11|3-HSD2 expression in primary
trophoblastic cells [14]. 11B-HSD2 gene expression in
human placental trophoblasts grown in primary culture
has been shown to maintain the same pattern as in vivo
[55] and dexamethasone stimulation regularly results in
an increase in 11B-HSD2 protein expression in tropho-
blasts [56]. Upon the finding that CRH induces 11(3-
HSD2 expression one cannot draw conclusions about
the activity of placental glucocorticoid metabolism. Inter-
estingly, Friedberg et al. [57] found a CRH-induced
reduction of llfi-HSDl activity in human adipocytes
in vitro. In isolated cytotrophoblasts Sharma et al. [58]
were unable to induce 118-HSD2 activity using CRH
concentrations of 1-100 ng/ml, however, they were able
to identify the CRH downstream signalling protein
p38MAPK [47] as an essential regulator for 11R-HSD2
activity. The fact, that we observed expressional changes
of lip~HSD2 following CRH treatment at much higher
CRH dosages (2.0 ug/ml) could however imply a possibil-
ity of a CRH-driven glucocorticoid induced feed-forward
mechanism on 11B-HSD2 activity. Although such a
mechanism has not been described for the placenta yet,
the subsequent reduction of Cortisol availability might be
an intriguing regulatory function shielding the fetus of
placental CRH-induced maternal glucocorticoids.

Our study focused on the auto- and paracrine effects
of CRH on leptin production in isolated trophoblasts,
as the placenta co-expresses both leptin (ObR-L) [59]
and CRH receptors [9]. We were able to show a sig-
nificant increase in leptin expression in syncytialised
trophoblastic cells following CRH treatment. While the
exact auto- and paracrine mechanisms and the func-
tional role of the interaction of CRH and leptin at the
level of the syncytiotrophoblast remain to be determined,
an increase of endocrine CRH and leptin expression
might translate into endocrine signals affecting both
fetus and mother, besides their local influence on
the trophoblast.

In this respect it is noteworthy, that the major frac-
tion of placental leptin and CRH is secreted into the

maternal circulation [60,61]. Nevertheless, the syncytio-
trophoblast is also involved in the maintenance of fetal
leptin and CRH serum levels [62,63]. Besides its role in
fetal organ maturation via Cortisol induction, there is in
fact evidence, that placental CRH drives parturition via
induction of adrenal DHEA-S on the fetal side followed
by an increase in placental estrogen secretion [63] .

In IUGR, a condition characterised by increased fetal
serum CRH levels [25], we found unchanged leptin
levels in fetal umbilical cord blood [23], despite an ele-
vated placental leptin mRNA and protein expression
[28,64]. Hence, it seems likely that CRH and CRH-
induced leptin (as suggested by our results) might inter-
act on the maternal side.

White et al. [65] showed that leptin has lipolytic
effects in rat placental tissue in vitro. CRH antagonises
lipolysis via down-regulation of llfi-HSDl in adipose
tissue [57]. Hypothetically leptin and CRH might act to-
gether in regulating the maintenance of fetal nutrient
supply at the placental level.

Conclusions

In summary, our data indicate that CRH stimulation
induces leptin secretion in the human syncytiotrophoblast
in an auto-/paracrine fashion. Similarly, CRH induced
11B-HSD2 expression. This suggests a short-loop feed-
back of CRH-induced leptin on CRH action at the feto-
maternal interface. Such a putative cross-talk could play
an essential role in the regulation of syncytiotrophoblast
nutrient supply and Cortisol metabolism, besides possible
further implications for myometrial contractility, placental
bed perfusion and the timing of birth. Furthermore CRH-
induced HB-HSD2 might locally determine placental
corticosteroid metabolism and thereby the passage of
placental CRH-triggered maternal Cortisol via the syncyt-
ium to the fetus. This would protect the fetus from
detrimental elevated maternal glucocorticoid exposure.
The underlying mechanism and the functional role of
the interaction of CRH with leptin and llfi-HSD2 at the
syncytiotrophoblast remain to be determined.

Competing interests

The authors declare that they have no competing interests.
Authors' contributions

FBF contributed to conception and design of the study, analysed and
interpreted the data and drafted the manuscript MR performed the cell
culture experiments, including RT-PCRs. GV performed ELISA analysis. RO
contributed to acquisition and analysis of the data, AH contributed to
interpretation of data and was involved in drafting the manuscript, MR and
CM-C were involved in the analysis of data and critically revised the
manuscript for important intellectual content, RS contributed to the
acquisition and analysis of data, WR critically revised the manuscript for
important intellectual content, JD contributed to the conception and design
of the study and critically revised the manuscript for important intellectual
content. All authors have given the final approval of the version to be
published.

Fahlbusch et al. Reproductive Biology and Endocrinology 2012, 10:80
http://www.rbej.eom/content/1 0/1 /80

Page 8 of 9

Acknowledgements

The authors thank Novo Nordisk (Investigator initiated research grant) and
the "Deutsche Forschungsgemeinschaft" (STR 923/1-1) for financial support.
We thank Ms. Bitterer and Mr. Wachtveitl for technical assistance. We also
thank the research staff of the Department of Gynecology and Obstetrics,
University of Erlangen, especially Mrs. Oeser, for their collaboration. The study
sponsors had no involvement in the collection, analysis and interpretation of
data, in the writing of the manuscript and in the decision to submit the
manuscript for publication.

Author details

'Department of Pediatrics and Adolescent Medicine, University of
Erlangen-Nurnberg, Erlangen, Germany, department of Gynecology and
Obstetrics, University of Erlangen-Nurnberg, Erlangen, Germany. 3 Childrens'
and Adolescents' Hospital, University of Cologne, Cologne, Germany.

Received: 13 January 2012 Accepted: 4 September 2012
Published: 12 September 2012

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doi:1 0.1 1 86/1 477-7827-1 0-80

Cite this article as: Fahlbusch et al: Corticotropin-releasing hormone
stimulates expression of leptin, 1 1 beta-HSD2 and syncytin-1 in primary
human trophoblasts. Reproductive Biology and Endocrinology 201 2 1 0:80.